The present invention relates generally to factors associated with inactivation of tumor suppressor proteins. More particularly, the present invention provides compositions and methods for screening of viruses for carcinogenic potential and screening of compounds for the capability of blocking the inhibitory effect of protein E6 on tumor suppressor protein p53.
Malignant transformation of cells has been linked to expression of proteins encoded by oncogenes (Huang et al., Cell, 39:79-87 (1984)). Cells may also be transformed by suppression of growth- and replication-inhibiting factors. Suppression of these inhibiting factors may allow unrestrained cell replication and malignant transformation. Lane and Benchimol, Genes and Devel., 4:1-8 (1990). Human protein p53 is one such inhibiting factor.
Many lines of evidence point to the importance of protein p53 in human carcinogenesis. Mutations within the p53 gene are the most frequent genetic aberration thus far associated with human cancer (Vogelstein, Nature, 348:681-682 (1990)) and individuals with germ line p53 mutation have an elevated risk of developing cancer (Malkin et al., Science, 250:1233-1238 (1990); Srivastava et al., Nature, 348:747-749 (1990)). The mutations identified in cancers are generally point mutations which fall within evolutionarily conserved domains and most of these mutated alleles have transforming activity in various cell culture assays (reviewed in Lane and Benchimol, supra and Levine et al., Nature, 351:453-456 (1991)).
Although p53 was originally classified as an oncogene, subsequent studies have shown that wild-type p53 actually has growth suppressive and tumor suppressive properties (Finlay et al., Cell, 57:1083-1093 (1989); Eliyahu et al., Proc. Natl. Acad. Sci. USA, 86:8763-8767 (1989)). Overexpression of wild-type p53 in normal cells or in transformed cells leads to growth arrest at the Gl/S border of the cell cycle (Diller et al., Mol. Cell. Biol., 10:5772-5781 (1990); Baker et al., Science, 249:912-915 (1990); Martinez et al., Genes and Devel., 5:151-159 (1991)). p53 has been shown to have negative effects on the transcription of various genes (Ginsberg et al., Proc. Natl. Acad. Sci. USA, 88:9979-9983 (1991)), as well as to act as a DNA-binding transcriptional transactivator (Kern et al., Science, 256:827-830 (1992); Funk et al., Mol. Cell. Biol., in press (1992); Farmer et al., Nature, 358:83-86 (1992)).
p53 was originally identified as a protein that co-immunoprecipitated with large T antigen from SV40 transformed cells (Lane and Crawford, Nature, 278:261-263 (1979); Linzer and Levine, Cell, 17:43-52 (1979)). It was subsequently shown that the ElB 55 kD protein of adenovirus 5 and the E6 protein of human papillomavirus (HPV) types 16 and 18 can also associate with wild-type p53 (Sarnow et al., Cell, 28:387-394 (1982); Werness et al., Science, 248:76-79 (1990)). It is believed that the interaction of these viral proteins with p53 aids in releasing infected cells from a block in the cell cycle, resulting in the replication of both the cellular and viral genomes. Additional cellular proteins are involved in transformation as mediated by these viruses, the best characterized being the retinoblastoma tumor suppressor protein (pRB). SV40 large T antigen, the adenovirus ElA proteins, and the anogenital-specific HPV E7 proteins each bind to pRB (Whyte et al., Nature, 334:124-129 (1988); DeCaprio et al., Cell, 58:1085-1095 (1988); Dyson et al., Science, 243:934-937 (1989); Munger et al., EMBO J., 8:4099-4105 (1989)).
The E6 oncoproteins of the cancer-associated or "high risk" human papillomaviruses (HPVs) target cellular p53 protein. The association of E6 with p53 leads to the specific ubiquitination and degradation of p53. This suggests that E6 deregulates cell growth control by eliminating the p53 tumor suppressor protein. Complex formation between E6 and p53 is required for degradation of p53. An additional cellular factor, designated E6-Associated Protein ("E6-AP"), which has a native and subunit molecular mass of approximately 100 kd is necessary for E6-p53 complex formation. Huibregtse et al., EMBO J., 13:4129-4135 (1991).
The HPVs that infect the anogenital tract can be classified as either "high risk" or "low risk" based on their association with cancer. HPV types 16 and 18 are the most common of the high risk group, while HPV type 6 and 11 are among the low risk types. Approximately 90% of cervical cancers contain HPV DNA of the high risk types, and these same DNAs are found in the precancerous epithelial lesions (zur Hausen and Schneider, "The Role of Papillomaviruses in Human Anogential Cancer", in The Papovaviridea, Vol. 2, Plenum, New York, 1987; Riou et al., Lancet, 335:1171-1174 (1990)). The low risk types are associated primarily with benign lesions such as condyloma acuminata and are only rarely found associated with cancers. Transfection of DNA of the high risk HPVs results in the extended life span and immortalization of primary human keratinocytes and fibroblasts in cell culture, whereas DNA of the low risk types does not (Durst et al., Oncogene, 1:251-256 (1987); Pirisi et al., J. Virol., 61:1061-1066 (1987); Schlegel et al., EMBO J., 7:3181-3187 (1988)). Mutational analyses have shown that the E6 and E7 genes of the high risk HPVs are both necessary and sufficient for this activity in keratinocytes and fibroblasts (Hawley-Nelson et al., EMBO J., 13:4129-4135 (1989); Munger et al., J. Virol., 63:4417-4421 (1989); Watanabe et al., J. Virol., 63:965-969 (1989)). The specific interactions of the E6 and E7 proteins with p53 and pRB, respectively, correlate with the high and low risk classification. The high risk HPV E7 proteins bind to pRB with a higher affinity than the low risk HPV E7 proteins (Munger et al., EMBO J., supra), and only the high risk HPV E6 proteins form detectable complexes with p53 in vitro (Werness et al., supra).
A striking difference between HPV immortalized cells and adenovirus 5 or SV40 immortalized cells is that the p53 levels are low in HPV containing cells (Scheffner et al., Proc. Natl. Acad. Sci. USA, 88:5523-5527 (1991)) but greatly elevated in Ad5 and SV40 immortalized cells (Oren et al., Mol. Cell. Biol., 1:101-110 (1981); Reich et al., Mol. Cell. Biol., 3:2143-2150 (1983)). The low p53 levels in HPV immortalized cells may be explained by the observation that complex formation between E6 and p53 in an in vitro rabbit reticulocyte system leads to the ubiquitination and proteolytic degradation of p53 (Scheffner et al., Cell, 63:1129-1136 (1990)). ElB (55 kd) and SV40 large T, on the other hand, sequester p53 into stable complexes (Oren et al., supra; Reich et al., supra). In each case the effect of the viral oncoprotein is to functionally inactivate p53, which apparently leads to cellular proliferation.
Further evidence that the HPV E6 and E7 proteins functionally inactivate the p53 and pRB gene products comes from studies that have examined the state of the p53 and pRB genes in HPV-containing and HPV-negative cervical carcinoma cell lines (Scheffner et al., supra, 1991; Crook et al., Oncogene, 6:873-875 (1991); Wrede et al., Mol. Carcin., 4:171-175 (1991)). HPV-containing cell lines were found to express wild-type p53 and pRB, whereas cell lines lacking HPV DNA contained mutations within both the p53 and RB genes. This indicates that inactivation of the p53 and pRB gene products is an important step in cervical carcinogenesis, and that this can occur either by mutation or as a consequence of their interaction with the HPV E6 and E7 proteins.
A means to inhibit degradation of p53 would provide an approach to block the carcinogenic potential of HPVs which inactivate p53 by targeting its degradation. Further, identification of the E6 of a "high risk" HPV in tissue samples would also provide a means for detecting high risk HPV in biopsy samples. What is also needed in the art are means of rapidly and specifically identifying the presence of high risk HPV and of blocking the effects of high risk E6. Quite surprisingly, the present invention fulfills these and other related needs.